1. Field of the Invention
The current invention relates to the use of a fusion protein ligand to couple with an antibody species to target recombinant viruses to specific cell or tissue of interest. The current invention can be used in viral vector based gene therapy. In the use for gene therapy, current invention broadens the spectrum of diseases amenable to gene therapy using viral vectors, enhances the viral transfection efficiency in cells or tissues that are refractory to the viruses, and finally provides a safer and more flexible system for gene targeting. Current invention can also be used in experimental setting to selectively transfect specific cells or tissues of interest in a mixed cell or tissue environment.
2. Description of Prior Art
Infectious microorganism, especially those by virus, is characteristically tissue and/or species specific. This characteristic is named viral tropism. It has been known that the tropism is mainly associated with the fact that viral entry requires interactions between viral surface proteins and cellular surface receptors and, in some case, also cellular coreceptors and the fact that the viral receptors are expressed in a tissue and/or species specific manner. The tropism, however, presents a limitation in the ability to use viral vectors for gene therapy if intended target cells are not viral native host cells. It is difficult to transfect recombinant viruses to cells lacking these receptors. Therefore, studies of viral receptor expression, receptor-virus interaction and mechanism of viral entry is very important to gene therapy research.
In the case of adenovirus, infection by adenovirus requires binding of viral fiber protein to the extracellular domains of a recently identified 46-kD membrane protein named coxsackievirus/adenovirus receptor (CAR) (Bergelson, et al., 1997, Science 275, 1320-3.). Fiber is a trimer with a structure similar to a knob. The interaction between CAR and viral fiber is highly specific, and of high affinity (Louis, et al., 1994, J Virol 68, 4104-6, Bergelson, et al., 1998, J Virol 72, 415-9.). The structure of the fiber knob in complex with extracellular domain of CAR has been delineated recently (Bewley, et al., 1999, Science 286, 1579-83, Roelvink, et al., 1999, Science 286, 1568-71.). The identified binding site for CARs is on the side of the fiber knob with three CAR monomers bound per fiber knob trimer. The multivalency of CAR binding may contribute to the high efficiency of adenovirus infection. Following initial binding, the viral protein penton base binds via its Arg-Gly-Asp (RGD) motif to xcex1vxcex23 or xcex1vxcex25 integrins of cell membrane and this binding activates virus internalization via receptor-mediated endocytosis (Wickham, et al., 1993, Cell 73, 309-19.). Inside coated vesicles conformational changes of viral protein trigger the passage of adenovirus core particle through the cell membrane (Wickham, et al., 1994, J Cell Biol 127, 257-64, Wang, et al., 1998, J Virol 72, 3455-8.). Both high affinity binding by adenovirus and endocytosis events by the host cells are necessary for the adenoviral transfection to occur. Because of its ability to transfect a variety of quiescent tissues or cells and to maintain a long-term transgene expression adenovirus has been preferred over other available gene delivery systems for gene therapy. In fact, a majority of clinical trials currently underway use recombinant adenoviral vector based gene delivery systems.
Retroviruses are another example in which presence of viral receptors on host cells are critical for viral entry. Retrovirus infects cells in a two step mechanism. These viruses contain two envelope glycoprotein subunits designated surface (SU) and transmembrane (TM) which form an oligomeric complex on the viral surface and mediate viral entry. The SU protein contains the viral receptor binding determinants whereas the TM protein contains a hydrophobic transmembrane region and a separate hydrophobic segment that mediates virus-cell membrane fusion (Weiss, 1993, The Retroviridae 2, 1-107.). The first step of infection is the attachment of the viral particle via the surface protein of the retrovirus envelope (env) protein and that is followed by viral and cellular membrane fusion for viral uptake. The env protein is largely responsible for the tissue or species specificity of the retroviral infectivity. In the infection by human immunodeficiency virus (HIV), the soluble cell surface receptor is CD4 membrane protein. While G protein-coupled chemokine receptors, such as CXCR4 or CCR5, each acts as coreceptors for the syncytium-inducing T-cell tropic X4 strains (Feng, et al., 1996, Science 272, 872-7.) and primary non-syncytium-inducing macrophage tropic R5 strains, respectively (Deng, et al., 1996, Nature 381, 661-6.). It is also evident that many primary HIV isolates are in fact dual tropic, having the ability to utilize both CXCR4 and CCR5 as coreceptors, and are named as R5X4 isolates.
Another example is adeno-associated viruses (AAV). AAV has a linear single-stranded DNA and only undergo productive infection if the infected cells are co-infected with a helper virus (e.g., adeno- or herpesvirus) otherwise the genome becomes integrated in a latent state at a specific site on a human chromosome (Bems, 1996, Fields Virology.). Recombinant adeno-associated viruses are typically made by replacing viral genes with desired genes of interest or by simply adding the terminal AAV DNA sequences (ITRS) to these genes. In the case of type 2 AAV, membrane associated heparan sulfate proteoglycan is a receptor for viral infection (Summerford and Samulski, 1998, J Virol 72, 1438-45.).
Other examples include negative strand RNA viruses. These viruses infect cells by a variety of different mechanisms. For example, Influenza A viruses which have a segmented RNA genome, contain a surface hemagglutinin protein which binds to cell surface sialic acid receptors and mediates viral entry in a low pH endosome following receptor-mediated endocytosis (Lamb and Krug, 1996, Fields Virology.). The positive strand RNA viruses also infect cells by receptor mediated entry. For example, among the picomaviruses, different members of the immunoglobulin protein superfamily are used as cellular receptors by poliovirus, by the major subgroups of rhinoviruses, and by coxsackie B viruses, whereas an integrin protein is used by some types of ecoviruses and a low density lipoprotein receptor is used by minor subgroups of rhinoviruses (Rueckert, 1996, Fields Virology.). Following receptor binding, it is not yet fully understood what role receptor-mediated endocytosis plays for picomaviral entry, if indeed it is required. Paramyxoviruses containing a non-segmented RNA genome have two surface viral proteins, the hemagglutinin (HN) and fusion protein (F), required for viral entry which occurs at neutral pH. These viruses utilize sialic acid receptors, or protein receptors, such as CD46 used by measles virus, for viral entry (Lamb and Kolakofsky, 1996, Fields Virology.). Rhabdoviruses (e.g., VSV) also have a non-segmented RNA genome, contain a surface protein (G) which also binds to specific cell surface receptors and mediates viral entry in a low pH endosome. In some cases, however, a specific phospholipid, in steady of protein peptide, appears to be one of the receptors for VSV (Wagner and Rose, 1996, Fields Virology.). The herpesviruses which have large double-stranded DNA genomes, contain a number of surface glycoproteins involved in viral entry and utilize various cell surface receptors. For example, herpes simplex virus and cytomegalovirus entry involves binding to a heparin sulfate cell surface receptor and herpes simplex viruses use other proteins (e.g., HVEM) for viral entry (Montgomery, et al., 1996, Cell 87, 427-36.). In contrast, Epstein-Barr virus entry is initiated by binding to a completely distinct cell surface receptor, CR2 (Wolf, et al., 1993, Intervirology 35, 26-39.). Strategies have been described that allow one to engineer herpes simplex viruses, cytomegaloviruses and Epstein-Barr viruses as vectors for heterologous gene expression (Roizman, 1996, Proc Nail Acad Sci USA 93, 11307-12, Marconi, et al., 1996, Proc Natl Acad Sci USA 93, 11319-20.). Because the picornaviruses lack a surface lipid bilayer, their entry pathway does not involve fusion of a viral membrane with a host cell membrane. In contrast, the alphaviruses (e.g., Sindbis virus and Semliki virus) do contain a surface lipid bilayer. These viruses contain two (E1 and E2) surface proteins, and in some cases a third (E3) surface protein important for viral entry. These viruses use various cell surface receptors. For example, Sindbis virus can use a laminin receptor or other receptors and generally enter cells by a pH-dependent mechanism, following receptor-mediated endocytosis (Schlesinger and Schlesinger, 1996, Fields Virology.). A pseudotyped virus has the env protein from a first retrovirus of a desired specificity and core or structural proteins from a second virus (e.g. a second retrovirus, an orthomyxovirus or a rhabdovirus). Viral pseudotypes have been described (Landau, et al., 1991, J Virol 65, 162-9, Dong, et al., 1992, J Virol 66, 7374-82, Le Guern and Levy, 1992, Proc Natl Acad Sci USA 89, 363-7.). A pseudotyped virus can be targeted to specific cell-types for viral entry in using a receptor mediated process. Poxviruses have large double stranded DNA genomes and enter cells by a pH independent mechanism via receptors that remain to be defined (Moss, 1996, Fields virology.). Poxvirus vectors have been used extensively for the expression of heterologous recombinant genes and as vaccines (Moss, 1996, Proc Natl Acad Sci USA 93, 11341-8, Paoletti, 1996, Proc Natl Acad Sci USA 93, 11349-53.).
In summary, various viral species mentioned above gain entry into host cells through specific cellular membrane receptors. In some cases, the membrane receptors have been identified, i.e., CD4 for HIV, CAR for adenovirus, etc. In other cases, the specific membrane protein or peptide that serves as viral receptor for viral entry has not been identified.
Recombinant adenoviral vectors are generated by a variety of techniques that include introducing a desired gene of interest into a bacterial plasmid at a site flanked by sequences that provide control elements for gene expression. These sequences are further flanked by DNA sequences from adenovirus. These sequences from adenovirus serve as sites for recombination with a compatible adenoviral genome when co-transfected together into an appropriate mammalian cell line (Horwitz, 1996, Fields Virology.).
However, to be safely and effectively used in gene therapy it is necessary to increase the viral transfection efficiency and selectivity. This is also true for adenoviral based gene delivery due to a broad low-level, non-uniform expression of CAR (Wickham, et al., 1996, J Virol 70, 6831-8.). Two major approaches have been underway to increase the transfection efficiency and selectivity. The first approach has been to modify the viral fiber protein by fusing specific peptides to the viral fiber. For example, a stretch of peptides specific for high affinity integrin xcex1v binding or heparan sulfate-containing receptor binding were inserted into fiber protein (Wickham, et al., 1997, J Virol 71, 8221-9.). These modifications significantly increased the viral transfection efficiency in malignant glioma cell lines (Staba, et al., 2000, Cancer Gene Ther 7, 13-9.). Similarly, fusing a peptide from adenovirus serotype 35 to the adenovirus serotype 5 capsid protein also increased viral transfection efficiency in CD34+ hematopoietic stem cells (Shayakhmetov, et al., 2000, J Virol 74, 2567-83.). Although these approaches have increased transfection efficiency of recombinant adenovirus in certain tissue, they generally widen rather than narrow the viral tropism because they do not block the viral transfection into its native hosts. In fact, these approaches by their nature are limited in their ability to modify fiber protein because conformational changes in fiber protein may affect its binding to CAR and subsequent viral propagation. The second approach has been to use a protein ligand to re-direct the recombinant adenovirus to selected tissue. In one of such experiment, an adenovirus neutralizing antibody was fused with the epidermal growth factor (EGF) (Watkins, et al., 1997, Gene Ther 4, 1004-12.) and the fusion protein re-directed recombinant adenovirus to EGF receptor-expressing cells. The limitation of this approach is its ability to be adapted for broad applications to other cell markers since not all surface markers have their native ligands. A cross-linking technique was also described in which viral particles were linked to specific antibodies (Rogers, et al., 1997, Gene Ther 4, 1387-92.). However, these approaches failed to block the infection of native host cells by recombinant adenovirus. Using bispecific antibodies which recognized both a FLAG tag inserted on the penton base of adenovirus and xcex1v integrin (or E-selectin) expressed on host cells, recombinant adenovirus particles were re-directed specifically to xcex1v integrin (or E-selectin) expressing cells (Wickham, et al., 1997, Cancer Immunol Immunother 45, 149-51.). This strategy also did not block the native host infection.
In the effort to target retrovirus in gene therapy, similarly strategy using a fusion protein ligand has also been reported recently. In one of such approach that is similar to the use of antibody fusing to EGF as a bispecific ligand to link virus to EGF receptor (Watkins, et al., 1997, Gene Ther 4, 1004-12.), the viral cellular receptor for retrovirus was used in replacement of an antibody species to be fused to EGF (Snitkovsky and Young, 1998, Proc Natl Acad Sci USA 95, 7063-8.). This fusion protein also demonstrated ability to target retrovirus to EGF expressing cells (Snitkovsky and Young, 1998, Proc Natl Acad Sci USA 95, 7063-8.). This finding was awarded a U.S patent (U.S. Pat. No. 6,060,316). However, like in the case to target recombinant adenovirus, employ EGF in fusion protein in targeting lacks flexibility. It also is limited to only EGF receptor expressing cells. Although EGF receptor is highly expressed on some tumor cells normal cells also express this receptor. Therefore, this strategy is not highly selective and not specific enough for safety reason if recombinant virus carrying a cytotoxic gene is used.
It would be highly desirable to be provided with means to block the native viral infection and target the virus with high selectivity so that when recombinant viral based vectors are used in clinical or experimental settings nonspecific and undesired viral infection does not occur.
It would be highly desirable to be provided with means to re-target recombinant viruses specifically to desired tissue with high efficiency so that the viral titer in gene delivery that is required to achieve therapeutic value is reduced in clinical or experimental settings. This step can be especially beneficial because reduced viral titer can reduce adverse effects.
It would also be highly desirable to be provided with means to target recombinant viruses with great flexibility and in a easily adaptable manner so that the targeting system can be adaptable to many different tissue or cell of interest and applicable in many conditions under clinical and experimental settings. This feature can be especially useful in broad applications.
The current invention relates to a novel targeting method for gene delivery by recombinant viruses. The recombinant viruses are targeted to any cells by a fusion protein ligand in coupling with a specific antibody species. The specific antibody is a monoclonal antibody which recognizes a specific antigenic determinant on the surface of an antigen or a purified polyclonal antibody which recognizes many different antigenic determinants on the surface of an antigen. In current targeting method, one end of the fusion protein ligand is the extracellular domain of a viral receptor that binds specifically to the surface of a virus while the other end is a IgG Fc-binding protein, such as protein A from bacteria, that binds specifically to the Fc region of an antibody. Antibody used is specific recognizing a cell surface marker that is present on the surface of target cells. The serial specific binding interactions, i.e., binding of the virus to fusion protein, fusion protein to antibody and antibody to cell surface marker, bring the recombinant viruses that contain a heterologous gene or genes encoding therapeutic protein(s) to the surface of target cell followed by viral entry in target cell.
In current invention, the interaction between a viral receptor and a viral species is specific (e.g., CAR binding to the fiber protein of adenovirus, and CD4 molecule binding to the gp120 protein of human immunodeficiency virus). The interaction between antibody and cell surface marker is also specific (e.g., anti ICAM-1 IgG binding to ICAM-1 molecules wherever it is expressed, and anti CD34 IgG binding to CD34 molecules). However since the Fc regions of all antibody species are structurally similar and can bind to the IgG Fc-binding protein, this feature makes the IgG Fc-binding protein of the fusion protein ligand capable to bind to any given antibody species. Because of this ability, fusion protein ligand in current invention can bind to different antibody species that recognizes different specific cell surface markers on different target cells. In this regard, current invention not only circumvents the requirement for expression of viral receptors on target cells but also circumvents the requirement for the presence of cellular receptors, such as EGF receptor (Snitkovsky and Young, 1998, Proc Natl Acad Sci USA 95, 7063-8.), on target cells, and co-presence of native peptide or protein ligands, such as EGF, for binding to the receptor (Snitkovsky and Young, 1998, Proc Natl Acad Sci USA 95, 7063-8.). In these prior arts targeting via the fusion proteins comprising EGF as a means to target EGF receptor expressing cells by Watkins for delivery of recombinant adenovirus (Watkins, et al., 1997, Gene Ther 4, 1004-12.) or by Snitkovsky for delivery of recombinant retrovirus ((Snitkovsky and Young, 1998, Proc Natl Acad Sci USA 95, 7063-8.) and U.S. Pat. No. 6,060,316), requires that EGF receptor is expressed on target cells in order for such delivery.
One aim of the present invention is to provide means to block the native viral infection and target the virus with high selectivity so that when recombinant viral based vectors are used in clinical or experimental settings nonspecific and undesired viral infection does not occur.
Another aim of the present invention is to re-target recombinant viruses specifically to desired tissue with high efficiency so that the viral titer in gene delivery that is required to achieve therapeutic value is reduced in clinical or experimental settings. This step can be especially beneficial because reduced viral titer can reduce adverse effects.
Another aim of the present invention is to target recombinant viruses with great flexibility and in a easily adaptable manner so that the targeting system can be adaptable to many different tissue or cell of interest and applicable in many conditions under clinical and experimental settings. This feature can be especially useful in broad applications.